Project Summary/Abstract Dendritic cells (DCs) are professional antigen presenting cells that bridge innate and adaptive immunity via the cross-presentation of antigens. DCs play an indispensable role in cancer immunity where they activate cytotoxic T cells to clear tumor cells. Despite this, cancers have developed methods to inactivate DCs in order to avoid immune clearance in a process called DC tolerization. In order to communicate with other cell types, cancer cells utilize 30-150 nm nanovesicles called exosomes. Exosomes released from the tumor transit to distant sites, including lymph nodes, where they promote the formation of a tumor supportive microenvironment called the ?pre-metastatic niche? by delivering specific RNAs and proteins. Due to the ability of exosomes to promote metastasis over long distances and the importance of DCs to tumor progression, it is likely that exosomes play a role in DC tolerization. Our laboratory has demonstrated that a DC fatty acid oxidation (FAO) metabolic program is a major determining factor in inducing tolerization. FAO-induced DC tolerization is characterized by the promotion of immunosuppressive cell types like regulatory T cells (Tregs) and by a reduced ability of DCs to present antigen. Preliminary data shows that tumor-derived exosomes are significantly taken up by DCs, both locally and in distant lymph nodes, but the effects of these exosomes remain unclear. We hypothesize that tumor-derived exosomes drive DC metabolism towards a FAO state resulting in an immunotolerant phenotype and cancer progression. First, we will use melanoma cell lines isolated from BRAFV600E and PTEN-/- transgenic mice that are engineered to express a CD63-RFP fusion protein to fluorescently tag melanoma exosomes. In tumor implant models, the biodistribution of exosomes to DCs and the resulting phenotypic changes of DCs in the lymph nodes, and primary tumor will be assessed using flow cytometry and RNA-seq. Next, we will utilize cellular metabolism analysis and high-resolution mass spectrometry to determine the impact of tumor-derived exosomes on changes in the flux of metabolites through key metabolic pathways. We anticipate that melanoma exosomes cause increased FAO leading to subsequent DC tolerization. Finally, the therapeutic application of utilizing oncolytic herpes viral vectors (oHSV1) to both kill tumor cells and suppress FAO in dendritic cells will be investigated. oHSV1 will be used to drive melanoma specific expression of either miR-33 or shRNA targeted to CPT1a preceded by a nucleotide motif (x-motif) targeting these small RNAs to exosomes. Both these RNAs target CPT1a an important mediator of FAO. Therefore, oHSV1 can be utilized to target DCs with mir33/shCPT1a containing exosomes, reduce FAO and promote an anti-tumor immune response while simultaneously killing tumor cells. Ultimately, these proposed studies will generate an improved understanding of the tumor-derived factors that drive dendritic cells towards an immunotolerant state.